The use of enzyme extracts from microorganisms in industry is widespread and profitable. Among the more common enzymes produced in a larger scale are bacterial proteases for use in making detergent powders, gluose oxidase for food preservation, and glucanases in the brewing industry. Many enzymes isolated for industrial use are extracellular, i.e., excreted into the growth medium by the microorganisms; isolation of such enzymes is usually a relatively simple matter. However, as is the case with, for example, glucose oxidase, many enzymes are produced intracellularly; extraction of the enzyme and removal of contaminants such as cellular debris and unwanted proteins presents an additional difficulty to the larger scale use of such products.
One particularly valuable intracellularly produced enzyme is glucose isomerase. This enzyme is produced by a wide variety of microorganisms, and is used to enzymatically catalyze the conversion of glucose, a relatively unsweet but inexpensive sugar to the sweeter sugar fructose. Examples of known procedures for this conversion may be found in Hamilton, et al. ("Glucose Isomerase, a Case Study of Enzyme-Catalyzed Process Technology" Immobilized Enzymes in Food and Microbial Processes, Olson, et al., Plenum Press, New York, (1974). pp. 94-106, 112, 115-137); and a number of other publications (Antrim, et al. "Glucose Isomerase Production of High-Fructose Syrups", Applied Biochemistry and Bioengineering, Vol. 2, Academic Press (1979); Chen, et al., "Glucose Isomerase (a review)", Process Biochem., (1980) 15(6), pp. 36-41; Thompson, et al. "Fructose Manufacture from Glucose by Immobilized Glucose Isomerase", Chem. Abstracts, Vol. 82, (1975), Abs. No. 110316h; and Takasaki, "Fructose Production by Glucose Isomerase", Chem. Abstracts, Vol. 81, (1974), Abs. No. 7647a)
Although the enzyme is water soluble, performing the reaction in an aqueous solution presents the difficulty and expense of recovering the enzyme; a single use of the enzyme may also be rather costly. There are therefore a number of techniques for isomerization which involve immobilizing the enzyme so that substantial activity is retained while the enzyme is fixed to a water insoluble matrix. This arrangement allows for the repeated use of the enzymes for prolonged periods of time and with a number of different glucose containing solutions.
For such a system to function at maximum efficiency the immobilized enzyme should preferably be as pure as possible. This allows not only maximum loading, but also provides maximum specificity during conversion by ensuring a homogeneous enzyme product. A number of types of purification methods now exist. U.S. Pat. No. 4,007,842 describes a method in which a water insoluble organic solvent is added to an aqueous solution of this enzyme, causing precipitation of non-enzyme material, followed by treating the remaining solution with a soluble magnesium salt, which then causes the precipitation of an enzyme-magnesium complex. While effective, the method described therein is timeconsuming and relatively expensive. U.S. Pat. No. 4,250,263 describes a system in which a crude glucose-isomerase composition is heat-treated to precipitate non-enzyme material, leaving a glucose isomerase containing solution. Although this method is somewhat simpler than that noted above, the relative purity of the heat-treated solution is not very high. U.S. Pat. No. 4,256,838 discloses a method in which glucose isomerase is purified by treating an enzyme containing solution with a reagent which will precipitate nucleic acids, followed by chromatographing the remaining solution on a cellulose column, and eluting the enzyme. This method is not only rather complicated, but also provides a resulting enzyme solution with a yield of only about 70% of the original enzyme activity.
The present invention teaches a method of glucose isomerase purification which provides a final enzyme containing solution of unexpectedly high yield and purity, utilizing a technique heretofore unknown for glucose isomerase purification. It involves a process of ultrafiltration of an enzyme extract in combination with a selective elution of the enzyme by use of a salt solution. The salt treatment of the enzyme retained on a membrane has the surprising effect of inducing permeation of the enzyme through the membrane which would not otherwise allow its passage; the mechanism by which this unexpected result is obtained is unknown. The resulting enzyme solution contains a yield of at least 75-80% of the original crude enzyme extract activity, and which is least 80% pure enzyme. Such a product is particularly well suited for immobilization on an appropriate support.